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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 <t>h,</t> <t>D-luciferin</t> (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .
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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 <t>h,</t> <t>D-luciferin</t> (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .
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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 <t>h,</t> <t>D-luciferin</t> (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .
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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 <t>h,</t> <t>D-luciferin</t> (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .
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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 <t>h,</t> <t>D-luciferin</t> (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .
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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 <t>h,</t> <t>D-luciferin</t> (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .
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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 <t>h,</t> <t>D-luciferin</t> (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .
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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 <t>h,</t> <t>D-luciferin</t> (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .
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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 <t>h,</t> <t>D-luciferin</t> (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .
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In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 h, D-luciferin (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .

Journal: Bioactive Materials

Article Title: GSH-responsive nanovaccine triggers immunogenic cell death and potent memory T cell immunity for durable, recurrence-free tumor eradication

doi: 10.1016/j.bioactmat.2026.04.025

Figure Lengend Snippet: In vitro evaluation of CDT-induced immunogenic cell death (ICD), dendritic cell (DC) maturation, and T cell activation . ( A ) Schematic illustration of CDT-triggered ICD and associated damage-associated molecular pattern (DAMP) release. ( B, C ) Bioluminescence images and quantitative analysis of extracellular ATP release from 4T1 cells after different treatments. ( D, E ) Quantitative analysis and CLSM images of HMGB1 release. ( F ) CLSM images showing CRT surface exposure in 4T1 cells after different treatments. ( G ) Schematic illustration of the co-culture system used to evaluate DC maturation and subsequent T cell activation. Immature DCs (iDCs) were isolated from bone marrow, and CD8 + T cells were isolated from the lymph nodes and spleens of naïve BALB/c mice. ( H, I ) Representative flow cytometric plots and quantitative analysis of mature DCs, defined as CD11c + CD80 + D86 + cells, after 24 h incubation with differently treated 4T1 cells, showing enhanced DC maturation in the SHINE group. ( J, K ) Representative flow cytometric plots and quantification of CD69 + cells among CD8 + T cells. ( L, M ) Representative flow cytometric plots and quantification of CD25 + cells among CD8 + T cells. ( N, O ) Quantification of granzyme B and perforin expression in CD8 + T cells, indicating enhanced T cell activation and effector function following SHINE treatment. ( P ) Schematic illustration of the in vitro tumor-killing assay. CDT-pretreated 4T1-Luc cells (CDT only) and CDT-pretreated 4T1-Luc cells co-cultured with effector CD8 + T cells at an effector-to-target (E:T) ratio of 5:1 were used to evaluate the additional contribution of immunotherapy. After 24 h, D-luciferin (150 μg mL −1 ) was added to quantify live tumor cells by luciferase activity. ( Q, R ) Bioluminescence images and quantitative analysis of surviving 4T1-Luc cells after treatment with CDT alone or combined CDT + immunotherapy at different concentrations, showing that addition of immunotherapy further enhanced tumor cell killing. M, MnO 2 @PEG; MR, MnO 2 @R848; MP, MnO 2 @aPD-L1; SHINE, MnO 2 @R848@aPD-L1. Data are presented as mean ± SD ( n = 3 ; n = 4 for (R)). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Panels A, G, and P created with BioRender.com .

Article Snippet: Tumor cell viability was assessed by adding D-luciferin (Caliper Life Sciences, Hopkinton, Massachusetts, USA; 150 μg mL −1 ) and quantifying bioluminescence using an IVIS Lumina XR system (PerkinElmer, Inc., Waltham, Massachusetts, USA).

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Isolation, Incubation, Expressing, Cell Culture, Luciferase, Activity Assay